The yeast Saccharomyces cerevisiae has been recognized as an especially favorable microorganism for practical applications of genetic engineering technology. The yeast cells, when transformed with a suitable gene-containing vector, can be made to efficiently synthesize the nucleic acid and protein products of the gene. However, a major limitation of this technology is the fact that the products synthesized by the yeast cells are not secreted into the medium. Therefore, the cells must be disrupted and the products separated from all of the cellular components. To avoid such a difficult and expensive separation, it has been proposed that the gene product in some way be coupled to a yeast polypeptide sequence that is normally secreted by the cell.
S. cerevisiae cells are known to secrete into the culture medium several proteins which are associated with the mating function of .alpha.-mating type (MAT.alpha.) and a-mating type (MATa) cells. Both of these phenotypes are mating, nonsporulating cells. MAT.alpha. cells product the peptide .alpha.-factor, which is secreted into the culture medium. MATa cells produce two secreted polypeptide products, a-factor and Barrier.
It has been postulated that the structural genes for one or more of the above yeast mating substances might be utilized as part of a fused gene producing a chimeric protein. This chimera would include the sequences that facilitate the processing and secretion of the desired protein product into the culture medium. The .alpha.-factor gene, which has been cloned, analyzed, and sequenced (Kurjan and Herskowitz ref.), has been proposed for this application.
Prior to the experimental work leading to the present invention the specific DNA sequence that codes for the Barrier protein was not known. The existence of the BAR1 gene had been deduced from genetic evidence, but such gene had not been isolated from the yeast genome.
Tests for determining the amount of .alpha.-factor activity and/or Barrier activity produced by yeast cells have been developed. The assays are based on the general principle that .alpha.-factor reversibly inhibits the vegetative growth of a-cells, and that Barrier activity reverses the .alpha.-factor inhibition, thereby permitting the a-cells to resume growth. Mutant strains of a-cells are available that do not secrete Barrier and are therefore abnormally sensitive to .alpha.-factor inhibition. These strains, which carry the bar1 mutation (also known as the sstl mutation), are particularly useful for these tests, and were utilized in the cloning of the BAR1 gene to be described. See, for example Duntze, MacKay and Manney (1970), MacKay and Manney (1974a,b), Manney, Jackson and Meade (1983); Manney (1983); Chan and Otte (1981), and Sprague et al. (1981).